U.S. patent number 11,306,364 [Application Number 16/928,164] was granted by the patent office on 2022-04-19 for tert promoter mutations in gliomas and a subset of tumors.
This patent grant is currently assigned to Duke University, The Johns Hopkins University. The grantee listed for this patent is Duke University, The Johns Hopkins University. Invention is credited to Chetan Bettegowda, Darell D. Bigner, Yuchen Jiao, Patrick J. Killela, Kenneth W. Kinzler, Nickolas Papadopoulos, Zachary J. Reitman, Bert Vogelstein, Hai Yan.
United States Patent |
11,306,364 |
Yan , et al. |
April 19, 2022 |
TERT promoter mutations in gliomas and a subset of tumors
Abstract
We surveyed 1,230 tumors of 60 different types and found that
tumors could be divided into types with low (<15%) and high
(.gtoreq.15%) frequencies of TERT promoter mutations. The nine
TERT-high tumor types almost always originated in tissues with
relatively low rates of self renewal, including melanomas,
liposarcomas, hepatocellular carcinomas, urothelial carcinomas,
squamous cell carcinomas of the tongue, medulloblastomas, and
subtypes of gliomas (including 83% of primary glioblastoma, the
most common brain tumor type). TERT and ATRX mutations were
mutually exclusive, suggesting that these two genetic mechanisms
confer equivalent selective growth advantages. In addition to their
implications for understanding the relationship between telomeres
and tumorigenesis, TERT mutations provide a biomarker for the early
detection of urinary tract and liver tumors and aid in the
classification and prognostication of brain tumors.
Inventors: |
Yan; Hai (Durham, NC),
Vogelstein; Bert (Baltimore, MD), Papadopoulos; Nickolas
(Towson, MD), Kinzler; Kenneth W. (Baltimore, MD), Jiao;
Yuchen (Columbia, MD), Bettegowda; Chetan (Lutherville,
MD), Bigner; Darell D. (Mebane, NC), Reitman; Zachary
J. (Durham, NC), Killela; Patrick J. (Durham, NC) |
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University
The Johns Hopkins University |
Durham
Baltimore |
NC
MD |
US
US |
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Assignee: |
Duke University (Durham,
NC)
The Johns Hopkins University (Baltimore, MD)
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Family
ID: |
1000006250897 |
Appl.
No.: |
16/928,164 |
Filed: |
July 14, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200399708 A1 |
Dec 24, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14765692 |
Jul 14, 2020 |
10711310 |
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PCT/US2014/016906 |
Feb 18, 2014 |
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61772249 |
Mar 4, 2013 |
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61766857 |
Feb 20, 2013 |
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61765909 |
Feb 18, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6886 (20130101); C12Q 2600/112 (20130101); C12Q
2600/156 (20130101) |
Current International
Class: |
C12Q
1/6886 (20180101); C07H 21/04 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1282422 |
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Jan 2001 |
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CN |
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2005304497 |
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Nov 2005 |
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JP |
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2004016813 |
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Feb 2004 |
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WO |
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|
Primary Examiner: Bausch; Sarae L
Attorney, Agent or Firm: Banner & Witcoff, Ltd.
Claims
The invention claimed is:
1. A method of identifying a mutation in a human subject and
treating a human subject, comprising: subjecting a nucleic acid
sample obtained from a tumor of the human selected from the group
consisting of: glioma, astrocytoma, oligodendroglioma, and
oligoastrocytoma, to a reaction whereby reaction products are
formed; detecting in the reaction products from the human tumor a
somatic mutation at nucleotide chr5 1,295,250 in hg19; and
administering to the subject with the somatic mutation a therapy
selected from the group consisting of chemotherapy, radiotherapy,
biological therapy, or surgery.
2. The method of claim 1, wherein the tumor is a glioma.
3. The method of claim 1, wherein the tumor is an astrocytoma.
4. The method of claim 1, wherein the tumor is an
oligodendroglioma.
5. The method of claim 1, wherein the tumor is an
oligoastrocytoma.
6. The method of claim 1, wherein the tumor is a primary
glioblastoma.
7. The method of claim 1, wherein the reaction comprises amplifying
the promoter or part of the promoter of a telomerase reverse
transcriptase (TERT) gene to form an amplicon.
8. The method of claim 7, wherein the amplicon is sequenced.
9. The method of claim 7, wherein the amplicon is hybridized to a
mutation specific oligonucleotide.
10. The method of claim 7, wherein the reaction employs
mutation-specific amplification primers.
11. The method of claim 1, wherein the reaction is a nucleic acid
hybridization reaction.
12. The method of claim 11, wherein the reaction employs a
mutation-specific hybridization probe.
13. The method of claim 1, wherein the reaction is a nucleic acid
sequencing reaction.
14. The method of claim 1, wherein the nucleic acid sample is
obtained from a primary tumor.
15. The method of claim 1, wherein prior to the step of subjecting,
nucleic acids are extracted from a primary tumor sample.
16. The method of claim 1, further comprising: subjecting a nucleic
acid sample obtained from a non-tumor tissue of the human to the
reaction; and confirming that the somatic mutation detected in the
tumor tissue is not in the nucleic acid sample obtained from the
non-tumor tissue.
17. The method of claim 1, wherein the therapy is chemotherapy.
18. The method of claim 1, wherein the therapy is radiotherapy.
19. The method of claim 1, wherein the therapy is biological
therapy.
20. The method of claim 1, wherein the therapy is surgery.
21. A method of detecting a mutation in a human subject and
treating a human subject, comprising: subjecting nucleic acid
samples obtained from a plurality of tumors of a plurality of
humans to a reaction to form reaction products wherein the
plurality of tumors are selected from the group consisting of:
glioma, astrocytoma, oligodendroglioma, and oligoastrocytoma;
detecting in the reaction products of at least one of the plurality
of nucleic acid samples obtained from the plurality of tumors a
somatic mutation at nucleotide chr5 1,295,250 in hg19; and
administering a therapy to at least one of the human subjects in
whose tumor the somatic mutation was detected, wherein the therapy
is selected from the group consisting of chemotherapy,
radiotherapy, biological therapy, or surgery.
22. The method of claim 21, wherein the nucleic acid samples
obtained from the plurality of tumors are obtained from primary
tumors.
23. The method of claim 21, wherein prior to the step of subjecting
nucleic acid samples obtained from the plurality of tumors, nucleic
acids are extracted from primary tumor samples.
24. The method of claim 21, wherein the tumors are gliomas.
25. The method of claim 21, wherein the tumors are primary
glioblastomas.
26. The method of claim 21, wherein the tumors are
astrocytomas.
27. The method of claim 21, wherein the tumors are
oligodendrogliomas.
28. The method of claim 21, wherein the tumors are
oligoastrocytomas.
29. The method of claim 21, wherein the reaction comprises
amplifying the promoter or part of the promoter to form an
amplicon.
30. The method of claim 29, wherein the reaction employs
mutation-specific amplification primers.
31. The method of claim 29, wherein the amplicon is sequenced.
32. The method of claim 29, wherein the amplicon is hybridized to a
mutation specific oligonucleotide.
33. The method of claim 21, wherein the reaction is a nucleic acid
hybridization reaction.
34. The method of claim 21, wherein the reaction is a nucleic acid
sequencing reaction.
35. The method of claim 21, wherein the therapy is
chemotherapy.
36. The method of claim 21, wherein the therapy is
radiotherapy.
37. The method of claim 21, wherein the therapy is biological
therapy.
38. The method of claim 21, wherein the therapy is surgery.
39. The method of claim 21, further comprising: subjecting a
plurality of nucleic acid samples obtained from a plurality of
non-tumor tissues of the plurality of humans to the reaction; and
confirming that the somatic mutation detected in the tumor tissue
of the at least one human subject is not present in the nucleic
acid sample obtained from the non-tumor tissue of the at least one
human subject.
Description
TECHNICAL FIELD OF THE INVENTION
This invention is related to the area of mutation detection. In
particular, it relates to mutations in non-coding regions of the
human genome.
BACKGROUND OF THE INVENTION
Telomeres are nucleoprotein complexes at the ends of eukaryotic
chromosomes that are required for chromosomal integrity. Several
hundred nucleotides of telomere repeats cap each chromosomal end,
and in the absence of telomerase activity, telomeres shorten with
each cell division (1). Eventually, uncapped telomeres trigger cell
death or senescence. Cancer cells seem to divide ad infinitum and
therefore, require some telomere maintenance mechanism to avoid
this fate. Because telomerase activity is generally higher in
cancer cells than normal cells, it was originally believed that
telomerase was somehow activated in cancer cells (2-6). However, it
was subsequently realized that telomerase was only inactive in
terminally differentiated cells and that normal stem cells in
self-renewing tissues retained telomerase activity (1, 7-9).
Because normal stem cells must replicate throughout the long
lifetimes of mammals (which can be more than a century in humans),
it is clear that such cells must also retain telomerase activity.
Because normal stem cells are thought to be the progenitors of
cancers, there would be no need to specifically activate telomerase
in cancer cells; the enzyme was already active in the precursors,
just as were the hundreds of other enzymes and proteins normally
required for cell proliferation.
This view was challenged by the discovery of another mechanism for
maintaining telomere length i.e., alternative lengthening of
telomeres (ALT) (10-12). ALT occurs in the absence of telomerase
activity and seems to be dependent on homologous recombination. It
occurs in a particularly high fraction of certain tumor types, such
as sarcomas, pancreatic neuroendocrine tumors, and brain tumors,
but rarely in most common tumor types, such as those tumor types of
the colon, breast, lung, prostate, or pancreas (13). Why would
cancer cells need ALT if telomerase activity was already
constitutively active in their precursors? This question was
highlighted by the discovery that many ALT cancers harbor mutations
in alpha thalassemia/mental retardation syndrome X-linked (ATRX) or
death-domain associated protein (DAXX), genes encoding proteins
that interact with each other at telomeres (10, 11). Presumably,
the absence of functional ATRX/DAXX complexes permits the
homologous recombination resulting in ALT. At minimum, these data
were compatible with the ideas that there could be a selective
advantage for genetic alterations that results in telomere
maintenance and that telomerase is not indefinitely activated in
all normal stem cell precursors of cancers.
Another challenge to the idea that genetic alterations were not
required for telomerase activation in cancer was raised by the
finding that mutations of the telomerase reverse transcriptase
(TERT) promoter occurred in .about.70% of melanomas and in a small
number of tumor cell lines derived from various tissue types (14,
15). Importantly, only 5 of 110 cell lines derived from lung,
stomach, ovary, uterus, or prostate cancers harbored TERT promoter
mutations, whereas 19 mutations were found among 37 cell lines
derived from various other tumor types. This situation is analogous
to the situation for ALT, which is infrequently observed in common
epithelial cancers but is observed more regularly in tumors derived
from nonepithelial cells, particularly sarcomas and brain tumors
(13).
There is a continuing need in the art for biomarkers that help
detect, monitor, and characterize tumors, as well as that help
predict the effects of tumors on patients.
SUMMARY OF THE INVENTION
According to one aspect of the invention a method is provided for
testing a nucleic acid sample of a human. A nucleic acid sample of
a human is tested and the presence of a somatic mutation in a
promoter of a telomerase reverse transcriptase (TERT) gene is
determined. The nucleic acid is from a cancer selected from the
group consisting of: a sarcoma, a hepatocellular carcinoma, urinary
tract cancer, a head and neck cancer, a medulloblastoma, a glioma,
an astrocytoma, an oligodenderoglioma, and an oligoastrocytoma.
Another aspect of the invention is a modified nucleic acid probe.
It comprises at least 18 nucleotides of a human TERT promoter. The
18 nucleotides include C228A or C229A.
Another aspect of the invention is a modified nucleic acid primer.
It comprises at least 18 nucleotides of a human TERT promoter. The
18 nucleotides include C228A or C229A.
These and other embodiments which will be apparent to those of
skill in the art upon reading the specification provide the art
with new tools for characterizing tumors and managing care of
cancer patients.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Frequency of TERT promoter mutations; 15 or more tumors
were analyzed in 26 tumor types. Gliomas are divided into primary
GBM, astrocytoma (including astrocytoma grades II and III, as well
as secondary GBM), and oligodendroglioma.
FIGS. 2A-2D. Mutations of selected genes in glioma subtypes. (FIG.
2A) Distribution of TERT mutations and other genetic events in 51
primary GBMs. (FIG. 2B) Distribution of TERT mutations and other
genetic events among 40 astrocytomas, including grades II-III
astrocytomas and grade IV secondary GBMs. (FIG. 2C) Distribution of
TERT mutations and other genetic events among 45
oligodendrogliomas. (FIG. 2D) Distribution of TERT mutations and
other genetic evens among 24 oligoastrocytomas. World Health
Organization tumor grade is indicated under each column. Blank box
denotes WT status in tumors.
FIG. 3. Survival of primary GBM patients with TERT promoter-mutated
tumors. Kaplan-Meier analysis of 50 primary GBM patients stratified
by TERT promoter mutational status. Patients with TERT promoter WT
tumors (n=13) survived longer than patients with TERT
promoter-mutated tumors (n=37); median survival was 27 mo among the
patients with TERT promoter WT tumors compared with 14 mo among
patients with TERT promoter-mutated tumors. The estimated hazard
ratio was 0.38 (95% confidence interval=0.18, 0.81; P=0.01, log
rank test).
DETAILED DESCRIPTION OF THE INVENTION
The inventors have developed assays that are useful for
characterizing tumors. The assays involve biomarkers which are
nucleic acid mutations, typically a single nucleotide mutation in a
non-coding region of chromosome 5. The mutations are located in the
promoter region of telomerase reverse transcriptase (TERT), at
particular sites, particularly at 1,295,228; 1,295,229; and
1,295,250 (hg19). Such mutations have the effect of increasing
expression of telomerase reverse transcriptase.
Biological samples which can be used in the present methods
include, but are not limited to, samples containing tissues, cells,
and/or biological fluids isolated from a subject. Examples of
biological samples include, but are not limited to, tissues, cells,
biopsies, blood, lymph, serum, plasma, urine, saliva, mucus and
tears. Biological samples may be tissue samples (such as a biopsy).
Biological samples may be obtained directly from a subject (e.g.,
by blood or tissue sampling) or from a third party (e.g., received
from an intermediary, such as a healthcare provider or lab
technician). Nucleic acids may be isolated and/or purified prior to
assaying. Nucleic acids are not obtained from cell lines grown in
culture for numerous generations, because such cell lines develop
mutations during the immortalization and culturing processes that
do not reflect in vivo tumorigenesis.
Nucleic acids can be assayed using techniques such as amplification
and hybridization and nucleic acid sequencing. Primers and probes
can be mutation specific. Alternatively, primers can be generic and
amplify a region that includes that potentially mutated region,
whether mutant or not. A second technique can then be used to
identify the mutation.
Mutations can be identified as somatic by comparing the tumor DNA
to DNA from a normal tissue of the human patient. Normal or control
DNA can be obtained from a tissue that is not involved in the
cancer. The presence of a mutation in the tumor DNA but not in the
control DNA indicates that the mutation is somatic, i.e., it is not
inherited.
Probes and primers which are used in the method can be have any
usable chemistry, so long as they have suitable base sequence and
function in the assay to specifically hybridize and/or prime
synthesis. Particular probes and primers may optionally have
chemistries that are not naturally occurring, such as modified or
alternative backbone chemistry, or additional moieties such as
labels or enzymes, or additional base sequences that are not found
in the TERT gene promoter region adjacent to the TERT gene
promoter-hybridizing sequences of the probes or primers. The
modified or alternative chemistries may enhance stability of a
nucleotide or enhance hybridization, for example, relative to
naturally occurring nucleic acids. Probes and/or primers may also
be bound to solid supports, such as arrays or beads.
Typically probes and primers are sufficiently long to achieve
specific hybridization to the desired target sequence without
spurious hybridization to other non-target sequences. Typically the
size of such nucleic acids is at least 12, at least 14, at least
16, at least 18, at least 20, at least 22, at least 24, at least
26, at least 28, or at least 30 nucleotides and less than 200, less
than 150, less than 125, less than 100, less than 75, less than 50,
or less than 40 nucleotides.
The high prevalence of the TERT promoter biomarkers in certain
cancers make them useful for detection of cancers at an early stage
and for following patients for evidence of progression or
recurrence once they have been diagnosed. Such detection and
monitoring can be done in samples such as plasma, cerebrospinal
fluid, and urine, and other body fluids or tissues. Tumors that are
the source of the analyte nucleic acids may be primary tumors or
metastases.
Once a TERT promoter mutation is identified in one of the test
nucleic acids, treatment or prophylaxis can be recommended and/or
administered. The presence or absence of TERT promoter mutation may
characterize the subtype of tumor or cell type source of the tumor
into a group of more similar tumors, providing refinement to the
recommended or prescribed treatment or monitoring plan. Suitable
treatments may involve watchful waiting, chemotherapy,
radiotherapy, biological therapy, surgery, or other suitable
management. Inhibitory agents such as antibodies and inhibitory RNA
molecules may also be used, once a TERT promoter mutation is
identified.
We formulated a hypothesis about the mechanisms responsible for
telomerase activity in cancers. We suggest that there are two ways
to maintain telomere lengths as cells divide: (i) through
epigenetic regulation of telomerase activity, which occurs in stem
cells of tissues that are rapidly renewing, and (ii) through
somatic mutations that maintain telomere lengths, such as mutations
in the TERT promoter or mutations in DAXX or ATRX. Those cancers
that originate in tissues that are constantly self-renewing, such
as cancers of the epithelia of the gastrointestinal tract and skin
or bone marrow, would be unlikely to harbor telomere-maintaining
mutations, because telomerase is already epigenetically activated
in their precursor cells. In contrast, tumors arising from cells
that are not constantly self-renewing, such as neurons, glial
cells, fibroblasts, hepatocytes, islet cells, and pancreatic ductal
epithelial cells, might frequently harbor such mutations. A
corollary of this hypothesis is that tumor types exhibiting high
frequencies of ALT would also exhibit high frequencies of TERT
mutations, and these mutations would be distributed in a mutually
exclusive fashion. To test these hypotheses as well as answer other
questions related to the role of TERT promoter mutations in various
cancer types, we determined the prevalence of TERT promoter
mutations in a large number of tumors.
The results described below, as well as the results published in
refs. 14 and 15, provide evidence that supports one of the
hypotheses raised in the Introduction and refutes others. The first
of these hypotheses was that TERT mutations would only be observed
in tumors derived from tissues that are not constantly
self-renewing under normal circumstances. This hypothesis was
supported in part: the vast majority of TERT promoter mutations
occurred in tumors derived from tissues that do not continually
self-renew. The TERT-H tumor types include only melanomas, certain
subtypes of glioma, medulloblastomas, squamous cell cancers of the
tongue, liposarcomas, HCCs, and urinary tract cancers. The normal
transitional cells of the urinary tract have very low proliferative
indices (0.64%.+-.0.52%), much lower than indices of
gastrointestinal tract, bone marrow, or skin (38). Normal
hepatocytes also do not turnover often (39), and glial cells are
thought to have limited capacity for self-renewal (40).
Two other observations also support the hypothesis. Pediatric
primary GBMs rarely contained TERT mutations (11%), whereas adult
primary GBMs frequently did (83%). Pediatric GBMs are presumably
derived from cells that are still dividing at the time of tumor
initiation, and therefore, there is no selective advantage
conferred by activating telomerase through a genetic mutation.
Adult GBMs, in contrast, are presumably derived from postmitotic
cells, and they should require telomerase activation. Similarly,
medulloblastomas are embryonal tumors that typically arise from
precursor cells with high self-renewal rates that do not usually
persist in adults. This finding is consistent with our observation
that the mean age of medulloblastoma patients with TERT mutations
was considerably older than the mean age of medulloblastoma
patients without TERT mutations.
There are, however, exceptions that belie the hypothesis that TERT
mutations occur only in non-self-renewing tissues. The epithelium
that lines the tongue constantly self-renews, but many squamous
carcinomas of the tongue harbored TERT mutations. Additionally, the
squamous epithelia of the tongue certainly would not be expected to
self-renew less than other squamous epithelia of the oral cavity,
but the latter rarely harbored TERT mutations. This finding may
suggest that squamous carcinomas of the tongue originate from a
different cell of origin than other oral cavity squamous
carcinomas. Conversely, only a subset of the tumor types derived
from non-self-renewing tissues was TERT-H. For example, the TERT-H
tumors included myxoid liposarcomas but not synovial sarcomas.
Moreover, cells of the pancreas (the islets of Langerhans and the
ductal epithelial cells) rarely renew, but pancreatic tumors of all
types (pancreatic neuroendocrine tumors, acinar carcinomas, and
pancreatic ductal adenocarcinomas) were all TERT-L. The most that
we can conclude at present is that non-self-renewing cell types are
the major sources of TERT-H tumors but that non-self-renewal is
only one of the factors that determines whether tumor cells with
TERT promoter mutations will have a selective growth advantage over
adjoining cells.
The first corollary to the hypothesis raised above was that tumor
types that displayed ALT would be those types that harbored TERT
promoter mutations. This corollary is soundly refuted by these
data, at least in general terms. Although tumor types of the CNS
and liposarcomas had high frequencies of ALT as well as high
frequencies of TERT promoter mutations, these tumor types were the
exceptions rather than the rule. For example, pancreatic
neuroendocrine tumors have very high frequencies of ALT but no
evidence of TERT mutations. Conversely, bladder cancers frequently
have TERT mutations but never have ALT (13). Additionally, even
among gliomas, pediatric GBMs have high frequencies of ALT and low
frequencies of TERT mutations, whereas adult GBMs have the reverse
pattern.
The second corollary was that the selective advantage afforded by
TERT mutation would be equivalent to the advantage afforded by ATRX
mutation (conferring ALT). This hypothesis was most effectively
tested in gliomas, in which both ATRX coding and TERT promoter
mutations were common. There was a striking mutual exclusivity with
respect to ATRX and TERT mutations (P<0.0001), lending strong
support to this idea.
These results also raise many unanswered questions. In some tumor
types, such as gliomas, we can imagine that all tumors have
genetically activated telomere maintenance programs through
mutations in either TERT or ATRX. However, in other tumor types
with frequent ATRX mutations, such as pancreatic neuroendocrine
tumors, what is responsible for activating telomerase in the
fraction of cases not exhibiting ALT if it is not a mutation in the
TERT promoter? Similarly, what is responsible for activating
telomerase in those tumors derived from non-self-renewing cell
types in which neither ALT nor TERT mutations is frequently
observed, such as synovial sarcomas or osteosarcomas? Also, there
are occasional individual tumors among the TERT-L types that have
TERT promoter mutations (e.g., cervical cancers, ovarian cancers,
and in ref. 15, lung cancers). What distinguishes these occasional
cancers from others of the same histopathologic subtype?
Whole-genome sequencing studies, rather than those studies limited
to the exome, might provide answers to these questions.
The results recorded here have practical as well as basic
scientific implications. Two-thirds of bladder cancers had TERT
promoter mutations, making it the most commonly mutated gene yet
identified in invasive urothelial carcinoma of the bladder. Given
the persistently high mortality rate despite multimodality
treatment in this group of patients, these mutations represent
ideal urinary biomarkers to detect bladder cancers at an early
stage and to follow patients for evidence of progression or
recurrence once they have been diagnosed (41). Similarly, the high
prevalence of TERT promoter mutations in HCCs and glioma subtypes
provides excellent candidate biomarkers for early detection (HCC)
or monitoring (HCC in the plasma and gliomas in the cerebrospinal
fluid) (42, 43).
Another practical implication involves diagnostics. We conjecture
that tumors with TERT promoter or ATRX mutations are derived from
different precursor cells and that either type of precursor cell is
different from those types that are the precursors of tumors
without such mutations. This distinction could aid classification
of the tumors in clinically meaningful ways. For example, FIG. 2
outline the major genetic alterations occurring in the three most
common types of gliomas. On the basis of the data in FIG. 2 A-C, we
speculate that oligodendrogliomas that lack TERT mutations but
contain ATRX mutations may behave more like astrocytomas than
oligodendrogliomas and vice versa. Similarly, the primary GBMs
without TERT mutations (15% of the total) may behave more like
advanced progressive astrocytomas, which generally lack TERT
mutations. This possibility is supported by the observation that
those primary GBM patients without TERT mutations had a longer
survival, on average, than other primary GBM patients (FIG. 3).
The above disclosure generally describes the present invention. All
references disclosed herein are expressly incorporated by
reference. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only, and are not intended to
limit the scope of the invention.
Example 1--Methods
All clinical information and tissue were obtained with consent and
Institutional Review Board approval from the various institutions
donating material to this study, and they were obtained in
accordance with the Health Insurance Portability and Accountability
Act. Tissue sections were reviewed by board-certified pathologists
to ensure that .gtoreq.50% of the cells used for DNA purification
were neoplastic and confirm histopathological diagnosis.
Oligonucleotides with the sequences 5'-M13-GGCCGATTCGACCTCTCT-3'
(SEQ ID NO: 1) and 5'-AGCACCTCGCGGTAGTGG-3' (SEQ ID NO: 2), where
M13 is a universal sequencing priming site with sequence
5'-tgtaaaacgacggccagt-3' (SEQ ID NO: 3), were used to PCR-amplify
the proximal TERT promoter containing C228 and C250 (chr5:
1,295,228; chr5: 1,295,250, respectively; hg19) for Sanger
sequencing using standard methods (44). Primary GBM copy number
data as well as ALT status were derived from the data published in
refs. 37, 45, and 46, and OTX2 copy number expression was derived
from the data published in ref. 27. Brain tumor patients were
treated at the Tisch Brain Tumor Center at Duke. For the purposes
of this study, secondary GBM designates a GBM that was resected
>1 year after a prior diagnosis of a lower-grade glioma (grades
I-III), and all other GBMs were considered to be primary GBMs.
Pediatric GBM samples were defined as those samples occurring
before 21 years of age.
Example 2
We attempted to evaluate at least 20 individual specimens of common
tumor types and fewer specimens of rare tumor types, depending on
availability of specimens in our laboratories. In those tumor types
in which our pilot studies showed a significant number of
mutations, additional tumors were evaluated. Melanomas and tumors
of the lung, stomach, and esophagus were excluded, because they had
already been adequately evaluated in the seminal papers cited (14,
15). When primary tumors rather than cell lines were used, we
ensured that the fraction of neoplastic cells was >50% through
histopathologic examination of frozen sections of the tissue blocks
used for DNA purification. In those cases in which the neoplastic
content was <50%, we microdissected the lesions to enrich the
neoplastic content to >50%. Primers were designed to amplify the
region containing the two TERT mutations that were previously
described--C228T and C250T--corresponding to the positions 124 and
146 bp, respectively, upstream of the TERT ATG start site (14, 15).
The PCR fragments were then purified and analyzed by conventional
Sanger sequencing.
In all, we evaluated TERT promoter mutations in 1,230 tumor
specimens and identified 231 mutations (18.8%) (Table 1). C228T and
C250T mutations accounted for 77.5% and 20.8% of the alterations,
respectively. Additionally, we detected four mutations that had not
been observed previously: three C228A mutations and one C229A
mutation. All four of these mutations as well as a representative
subset of the C228T and C250T mutations (n=59) were somatic, as
evidenced by their absence in normal tissues of the patients
containing the mutations in their tumors.
TABLE-US-00001 TABLE 1 Frequency of TERT promoter mutations No. No.
tumors Tumor type* tumors mutated (%) Chondrosarcoma 2 1 (50)
Dysembryoplastic neuroepithelial tumor 3 1 (33.3) Endometrial
cancer 19 2 (10.5) Ependymoma 36 1 (2.7) Fibrosarcoma 3 1 (33.3)
Glioma.dagger. 223 114 (51.1) Hepatocellular carcinoma 61 27 (44.2)
Medulloblastoma 91 19 (20.8) Myxofibrosarcoma 10 1 (10.0) Myxoid
liposarcoma 24 19 (79.1) Neuroblastoma 22 2 (9) Osteosarcoma 23 1
(4.3) Ovarian, clear cell carcinoma 12 2 (16.6) Ovarian, low grade
serous 8 1 (12.5) Solitary fibrous tumor (SFT) 10 2 (20.0) Squamous
cell carcinoma of head and neck 70 12 (17.1) Squamous cell
carcinoma of the cervix 22 1 (4.5) Squamous cell carcinoma of the
skin 5 1 (20) Urothelial carcinoma of bladder 21 14 (66.6)
Urothelial carcinoma of upper urinary 19 9 (47.3) epithelium *No
mutations were found in acute myeloid leukemia (n = 48), alveolar
rhabdomyosarcoma (n = 7), atypical lipomatous tumor (n = 10),
breast carcinoma (n = 88), cholangiosarcoma (n = 28),
central/conventional chondrosarcoma (n = 9), chronic lympoid
leukemia (n = 15), chronic myeloid leukemia (n = 6), colorectal
adenocarcinoma (n = 22), embryonal rhabdomyosarcoma (n = 8),
esthesioneuroblastoma (n = 11), extraskeletal myxoid chondrosarcoma
(n = 3), fibrolammellar carcinoma of the liver (n = 12), gall
bladder carcinoma (n = 10), gastrointestinal stromal tumor (n = 9),
hepatoblastoma (n = 3), leiomyosarcoma (n = 3), conventional lipoma
(n = 8), low grade fibromyxoid sarcoma (n = 9), malignant
peripheral nerve sheath tumor (n = 3), medullary thyroid carcinoma
(n = 24), meningioma (n = 20), mesothelioma (n = 4), pancreatic
acinar carcinoma (n = 25), pancreatic ductal adenocarcinoma (n =
24), pancreatic neuroendocrine tumor (n = 68), prostate carcinoma
(n = 34), spinal ependymoma (n = 9), synovial sarcoma (n = 16), or
undifferentiated pleomorphic soft tissue sarcoma (n = 10) samples.
.dagger.Glioma comprises 11 subtypes; see Table 2.
TABLE-US-00002 TABLE 2 TERT mutations in glioma subtypes No. of
tumors with No. of TERT Tumors with tumors promoter TERT Glioma
subtype WHO grade studied mutation mutation (%) Primary GBM, IV 78
65 83 adult Primary GBM, IV 19 2 11 pediatric Astrocytoma II 8 0 0
Astrocytoma III 27 4 15 Astrocytoma IV 5 0 0 Oligodendroglioma II
19 12 63 Oligodendroglioma III 26 23 88 Oligoastrocytoma II 9 2 22
Oligoastrocytoma III 15 4 27
The 1,230 tumors represented 60 tumor types. In 26 of these tumor
types, at least 15 individual tumors were evaluated (comprising a
total of 1,043 individual tumors) (FIG. 1). In the remaining tumor
types, only a small number of samples (2-12) was available, in part
because these tumor types are generally uncommon in Western
populations (Table 1). Among the tumor types in which at least 15
individual tumors were available for study, a clear distinction
could be made. Eighteen of these tumor types had only occasional
TERT promoter mutations (zero to three mutations, comprising 0-15%
of the tumors of each type) (FIG. 1). We classified these tumor
types as TERT-low (TERT-L), because they had a low frequency of
TERT promoter mutations. Eight other tumor types were classified as
TERT-high (TERT-H) because of their relatively high prevalence of
TERT promoter mutations (16-83% of the tumors of each type).
The TERT-L tumor types included some of the most prevalent cancers,
including epithelial tumors of the breast, prostate, thyroid,
pancreas, gall bladder, uterus, and colon (as well as tumors of the
lung, stomach, and esophagus based on prior studies) (14, 15) and
leukemias. In fact, no TERT mutations were identified in any
specimen of 30 tumor types that we studied, comprising a total of
546 tumors (Table 1). Some nonepithelial cancers, such as synovial
sarcomas, chordomas, neuroblastomas, osteosarcomas, and
ependymomas, were also TERT-L.
Eight TERT-H tumor types were identified (in addition to the
previously described melanomas) (14, 15). These tumors included
tumors of the CNS, transitional cell carcinomas of the urinary
tract, hepatocellular carcinomas, myxoid liposarcomas, and
oral cavity carcinomas. Although only a small number of TERT-H
tumors (other than melanomas) were examined in previous studies
(15), mutations in gliomas, hepatocellular, and oral cavity
carcinomas were detected, which would be expected on the basis of
the high frequency of mutation in these tumors types (Table 1).
Example 3
Sarcomas.
One of the highest frequencies of TERT promoter mutation was found
in myxoid liposarcoma (19 of 24 tumors, 79% with mutation). Myxoid
liposarcomas account for more than one-third of all liposarcomas
and .about.10% of all adult soft tissue sarcomas (16). Patients are
relatively young, with a peak age range between 30 and 50 y. At the
genetic level, the most characteristic change is a t(12; 16) (q13;
p11) chromosomal translocation that results in the fusion of the
FUS and DDIT3 genes (16, 17). The cellular origin of these tumors
is unknown, but preadipocytic progenitor cells and mesenchymal stem
cells have been implicated (18); after embryogenesis, the mitotic
activity of these cells is thought to be low. Other sarcomas, also
thought to originate from mesenchymal cells that do not self-renew
in the absence of damage, were not TERT-H (Table 1). These sarcomas
included synovial sarcomas (0% of 16 tumors) and osteosarcomas
(4.3% of 23 tumors). Of note, myxoid liposarcomas have been
previously shown to have a relatively high prevalence of ALT (24%
of 38 tumors) (13, 19). The data, in aggregate, are compatible with
the idea that myxoid liposarcomas almost always genetically
activate telomere maintenance genes through either TERT promoter
mutations or ALT.
Hepatocellular Carcinomas.
Hepatocellular carcinomas (HCCs) are the third leading cause of
cancer mortality worldwide, and their incidence is increasing in
the United States (20). Most HCCs in the United States are
associated with Hepatitis B or C Virus infection, whereas others
are associated with alcoholic cirrhosis; 44% of HCC samples that we
evaluated harbored TERT promoter mutations (27/61). This finding
makes TERT the most commonly mutated gene yet observed in this
tumor type (21, 22). The mutations seemed to occur relatively early
in tumorigenesis, because they were observed in 39% of stage I
well-differentiated HCCs (Table S1). TERT mutations were observed
in virally associated tumors as well as cases without any
underlying liver disease at similar frequencies (Table S1). There
was also no difference in the prevalence of TERT promoter mutations
with respect to sex, age, or ethnicity (Table S1). ALT has been
observed in 7% of 121 HCCs studied previously (13).
Urinary Tract Cancers.
Urothelial carcinoma of the bladder is the fourth most common type
of cancer in American males. In 2013, over 73,000 patients will be
diagnosed with bladder cancer leading to approximately 15,000
deaths in the US alone (23). Two-thirds of the 21 urothelial
carcinomas of the bladder that we studied harbored TERT promoter
mutations. We were also able to evaluate 19 urothelial carcinomas
of the upper urinary tract, a much less common anatomic site for
this histopathologic subtype of tumor. Nine of nineteen upper
urinary tract urothelial carcinomas harbored TERT mutations. TERT
mutations are, therefore, the most frequently mutated genes yet
identified in urothelial carcinoma of either the bladder or upper
urinary tract (24). The prevalence of ALT in bladder cancers is
very low (1% of 188 cancers) (13).
Head and Neck Cancers.
Head and neck cancers are almost always squamous cell carcinomas
and can occur throughout the oral cavity lining (mucous membranes
of the cheek, hard and soft palate, tongue, supraglottis, etc.). It
is the sixth most common cancer in the world, and 50,000 cases
occurred in the United States in 2012. We identified TERT promoter
mutations in 17% of 70 oral cavity cancers that we evaluated.
However, the anatomic distribution of the cases with TERT promoter
mutations was striking: 11 of 12 cancers with TERT promoter
mutations were in the oral tongue, although only 23 of 70 total
cases originated in the oral tongue (P<0.0001, Fisher exact
probability test, two-tailed) (Table S2). The basis for this
extraordinary selectivity is curious given the shared
characteristics of the squamous epithelium lining the tongue and
other parts of the head and neck, including the oral cavity.
Moreover, we evaluated 22 squamous cell carcinomas of another site
(the cervix) and found only one TERT mutation (4.5%) (Table 1).
Most cervical squamous cell carcinomas and a subset of head and
neck squamous cell carcinomas are caused by human papillomavirus,
which can activate telomerase by expressing E6 and E7 viral
oncogenes (25). These findings raise the possibility that human
papillomavirus infection and TERT mutation may be alternative
mechanisms to activate telomerase among squamous cell carcinomas.
We were unable to test correlations between TERT promoter mutations
and HPV status or other clinical parameters because of the small
number of patients with available data (Table S2). There have been
no ALT cases identified among 70 head and neck cancers, including
41 oral cavity cancers (13).
Medulloblastomas.
Medulloblastoma is the most common malignant brain tumor of
childhood (26). TERT mutations occurred in 21% of 91
medulloblastomas that we evaluated. As with the oral cavity
cancers, TERT mutations were not distributed randomly among the
medulloblastoma patients. Although medulloblastomas are usually
diagnosed at a young age, those medulloblastomas with TERT
mutations were diagnosed at a considerably older age (median=6 vs.
16 y, P=0.0012, t test assuming unequal variances, two-tailed)
(FIG. S1A). This observation has important implications for
understanding the basis for the selectivity of the tumor types
harboring TERT promoter mutations (Discussion); 45 of 90 patients
had been assessed previously for orthodenticle homeobox 2 (OTX2)
gene amplification and expression, and alterations in this
transcription factor are known to correlate with clinically
distinct molecular subtypes of medulloblastoma (27). OTX2
expression was >100-fold higher in medulloblastoma patients
without TERT promoter mutations than in those patients with TERT
promoter mutations (note the log scale in FIG. S1B). The high
levels of OTX2 expression were usually the result of OTX2 gene
amplification (FIG. S1C). The association of TERT promoter
mutations with an older age at diagnosis and a lack of OTX2
overexpression raises the possibility that TERT mutations occur in
a specific clinical and molecular subtype of medulloblastoma. The
most likely molecular subtype of medulloblastoma that may be
enriched for TERT mutations is the noninfant sonic hedgehog
subtype, which is characterized by an older age at diagnosis and
lower expression of OTX2 (28, 29). Larger studies will be needed to
make this association more definitive. ALT has been observed in 7%
of 55 medulloblastomas studied previously (13).
Gliomas.
Gliomas are the most common CNS tumor type and accounted for
>14,000 deaths in the United States last year (30).
Histopathological and clinical criteria established by the World
Health Organization are used to characterize these tumors into
several subtypes (30). We considered the four main subtypes
individually (Table S3).
Primary Glioblastoma.
These primary glioblastomas (GBMs) are the most common malignant
brain tumors in adults, accounting for .about.17% of all
intracranial tumors, and they confer the worst survival (median of
.about.15 mo) (31). These high-grade (grade IV) tumors have no
detectable precursor lesions and have been referred to as de novo
tumors. The prevalence of TERT promoter mutations was remarkably
high in GBMs of adults (83% of 78 tumors) (Table S3). This
prevalence is higher than the prevalence of any other genetic
mutation in this tumor type (32). These findings provide a
molecular mechanism responsible for the high levels of TERT mRNA
and telomerase activity observed in GBMs (33).
For 51 of 78 primary GBM tumors, data on other common genetic
alterations as well as clinical data were available (FIG. 2A).
Interestingly, EGFR amplification, a classic molecular feature of
primary GBM, exclusively occurred in tumors with TERT mutations
(P=0.0006, Fisher exact probability test, two-tailed). Conversely,
no association was identified between TERT mutation and either TP53
mutation or CDKN2A deletion. Importantly, the frequency of TERT
promoter mutations was considerably less in primary GBMs of
pediatric patients (11% of 19 tumors) than adult patients
(Discussion) (Table S3). ALT was observed in 11% of 105 adult GBM
and 44% of pediatric GBM (i.e., the reverse of the pattern observed
for TERT promoter mutations) (13). Primary GBM patients without
TERT mutations survived considerably longer, on average, than
patients with such mutations (median=27 vs. 14 mo, P=0.01 by the
log rank test) (FIG. S3).
Astrocytomas.
Infiltrative astrocytic tumors frequently progress, with recurrent
lesions often of higher grade than the original lesions excised at
surgery. They are most often grade II or III but can progress to
grade IV (at which point they are often termed secondary GBMs).
Astrocytomas of any stage rarely contained TERT promoter mutations
(10% of 40 total samples) (Table S3). Instead, they more frequently
contained isocitrate dehydrogenase 1 (IDH1) or isocitrate
dehydrogenase 2 (IDH2) mutations (75% of 40 tumors), ATRX mutations
(70% of 40 tumors), and TP53 mutations (73% of 40 tumors) (FIG.
2B). ALT has been observed in 63% of 57 astrocytomas, consistent
with the high prevalence of ATRX mutations (13). The lack of
activating TERT mutations in IDH1 mutant tumors is also
corroborated by the lack of TERT mRNA and telomerase activity
observed in these lesions (33).
Oligodendrogliomas.
Like astrocytomas, oligodendrogliomas often progress, and they
frequently contain TERT promoter mutations (78% of 45 tumor
samples) (Table S3). Oligodendroglioma was the only tumor type
studied (of all types, including non-CNS tumors) (Dataset S1) in
which C250T mutations were nearly as frequent as C228T mutations.
In oligodendrogliomas, 43% of tumors with TERT mutations contained
C250T substitutions, whereas in other gliomas, only 10% did
(P<0.001, Fisher exact probability test, two-tailed).
Interestingly, 91% of 45 oligodendrogliomas that were evaluated for
ATRX and TERT sequence alterations contained either an ATRX coding
or a TERT promoter mutation, suggesting that genetic alterations
resulting in telomere maintenance are required for tumorigenesis of
this subtype.
Oligodendrogliomas have long been known to contain characteristic
losses of chromosome arms 1p and 19q, and these losses reflect
inactivation of the CIC gene on chromosome 19q and in some cases,
inactivation of the FUBP1 gene on chromosome 1p (34-36).
Accordingly, 78% of 45 oligodendrogliomas contained chromosome arm
1p or 19q losses of heterozygosity (FIG. 2C) (34-36). Moreover,
nearly all of them contained IDH1 or IDH2 mutations (93%).
Oligoastrocytomas.
As their name implies, these tumors are mixed, with histologic
features of both oligodendrogliomas and astrocytomas. This mixture,
in part, reflects the difficulties in distinguishing the various
glioma subtypes from one another on the basis of histopathologic or
clinical criteria (37). The genetic features of this tumor subtype
reflect this mixture: the prevalence of TERT promoter mutations
(25% of 24 tumors) was intermediate between oligodendrogliomas and
astrocytomas, as were the frequencies of chromosome (Chr) 1p/19q
losses and IDH1/2, TP53, and ATRX mutations (FIG. 2D).
Example 4--ALT Vs. TERT
ALT has been observed in tumors of the CNS (particularly gliomas)
more frequently than tumors of any other tissue type. Given that
TERT promoter mutations are also common in gliomas, the
relationship between these two features could be determined with
high confidence. The tumors depicted in FIG. 2 had previously been
evaluated for alterations in ATRX, which is a nearly perfect
surrogate for the ALT phenotype (11, 37). Our data show that there
were 50 gliomas with ATRX mutations and 83 gliomas with TERT
mutations; 0 of 83 tumors with TERT mutations contained ATRX
mutations (P<0.0001, Fisher exact probability test,
two-tailed).
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References